Liver Agxt expression. (a) Northern blot of total liver RNA from two Agxt^+/+, three Agxt^+/−, and three Agxt^−/− mice probed with Agxt cDNA probe (Upper) shows lack of expression of the targeted allele and reduced levels in heterozygous animals. (Lower) Reprobing of the blot with β-actin gene cDNA detects relative underloading of the heterozygous samples, which might account for the less-than-half signal seen on the Agxt^+/− samples with the Agxt probe. However, the Agxt^−/− samples, with β-actin hybridization signal similar to that of the wild-type, show no Agxt mRNA detectable after 1 day of exposure of the blot. (b) (Upper) Western blot of 50 μg of liver protein from Agxt^+/+, Agxt^+/−, and Agxt^−/− mice probed with affinity-purified rabbit antibody raised against recombinant mouse AGT shows lack of expression of the targeted allele and reduced levels in the heterozygous sample. (Lower) Reprobing of the blot with rabbit anti-GAPDH detects even loading of the gel.

Localization of AGT and AGXT^LRM in subcellular fractions of Agxt^−/− mouse livers after adenovector-mediated gene transfer. (a) Western blot. H, human liver homogenate; L, liver homogenate from untreated Agxt^−/− mouse; 1, 2, and 3, liver homogenate and subcellular fractions from an Agxt^−/− mouse treated with Ad-AGXT. 1, liver homogenate; 2, peroxisome-enriched fraction; 3, mitochondria-enriched fraction. 4, 5, and 6, liver homogenate and subcellular fractions from an Agxt^−/− mouse treated with Ad-AGXT^LRM. 4, Liver homogenate; 5, peroxisome-enriched fraction; 6, mitochondria-enriched fraction. (b) AGXT activity in liver homogenate (H) and subcellular fractions enriched in peroxisomes (P) or mitochondria (M) from wild-type C57BL/6 mice (WT). Agxt^−/− mice and Agxt^−/− mice treated with Ad-AGXT or Ad-AGXT^LRM are shown as indicated (means ± SD, n = 6). (c) Catalase (peroxisomal marker) and succinate dehydrogenase (mitochondrial marker) activities in liver homogenate (H), and mitochondrial (M) and peroxisomal (P) fractions from Agxt^−/− mice are shown (n = 6). The specific activity of catalase in the peroxisomal fraction, relative to that in the liver homogenate, was ≈60, whereas the mitochondrial fraction was depleted to a relative specific activity of 0.8. Relative specific activity of succinate dehydrogenase was ≈6 in the mitochondrial fraction and was depleted to 0.2 in the peroxisomal fraction. (d and e) Transmission electron microscopy of fractions of the liver homogenate from an Agxt^−/− mouse. (d) Peroxisome (P)-enriched fraction. (e) Mitochondria (M)-enriched fraction.

Targeted mutagenesis of the mouse Agxt locus. (a) Genomic organization of the endogenous Agxt locus (Upper), with exons shown as numbered boxes. The homology regions of the 5′ and 3′ arms included in the targeting construct (Lower) are indicated. The location of 5′ and 3′ external probes used for screening are shown as thick short lines. The primers flanking one of the replacement junctions, used for genotyping by PCR, are depicted as short arrows. Restriction enzyme sites: B, BamHI; H, HindIII; N, NcoI; and R, EcoRI. (b) Genotyping of the induced Agxt mutation in ES clones by using Southern blotting with either the 5′ flanking probe and BamHI-treated DNA (Left) or the 3′ flanking probe and NcoI-digested DNA (Right). Double homologous recombination, resulting in the replacement of exons 4–8 with the Pgk-Neo cassette, is identified by the presence of a 3.2-kb BamHI and a 9.8-kb NcoI band, as seen in the clone labeled (∗). WT, wild-type fragments; KO, mutant fragments. (c) Genotyping of tail tip DNA by PCR analysis, using the wild-type-specific primer in exon 4 and the mutant-specific primer in the neoresistance cassette, together with a common forward primer complementary to exon 3. Wild-type and mutated alleles are detected in a three-primer PCR as 0.6-kb and 0.4-kb products, respectively.

Urinary system phenotype of AgtKO mice. (a) Kidney histology of one of the few Agxt^−/− mice that showed CaOx deposits (dark black spots) under normal conditions. The precipitates are present in the distal collecting ducts at the renal papilla. (b) Abundant CaOx deposits are seen both at the cortex and medulla of Agxt^−/− mice after 3 weeks of 0.7% EG administration. (c) Microscopic image of urine sediments from Agxt^−/− mice, with clusters of tetrahedric “envelope-like” crystals, characteristic of COD. (d) Stereo microscope view of a large, unique, urine bladder stone with sharp edges, which yielded x-ray spectra of COD (f). (e) View of multiple, smaller, opaque stones, which resulted in x-ray spectra of COM (g). Stones were photographed over a grid of 1-mm squares, and the spectra were superimposed with those of pure standards (COD in f and COM in g), shown as vertical lines along the spectra.